Multi-dimensional chromatographic system for analyzing multiple sample components

ABSTRACT

A chromatography system includes a first chromatography column for receiving and separating a flow stream, a plurality of traps configured to trap a plurality of distinct flow segments exiting the first chromatography column during separation of the flow stream, and a second chromatography column operatively associated with the plurality of traps for receiving and separating the distinct flow segments. The system can include at-column dilution at trapping and separating stages thereof. A chromatography method for operating the chromatographic system includes measuring a plurality of time segments corresponding to a plurality of peaks of a fluid sample flowing through the first chromatographic column, and sequentially fluidly coupling the plurality of distinct flow segments with the corresponding plurality of traps during time segments corresponding to the plurality of peaks.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/305,915 filed Mar. 9, 2016, and U.S.Provisional Patent Application No. 62/286,603, filed Jan. 25, 2016, thecontents of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates generally to chromatography systems andmethods for performing multi-dimensional chromatography. Moreparticularly, the present disclosure relates to a system and method forenhancing multi-dimensional chromatography during a single loading of afluid sample.

2. Description of Related Art

In many fields of science, purified compounds are required for testingand analysis protocols. Purification of a compound involves separatingout a desired component or components from a mixture that containsadditional components or impurities. Chromatography is a method offractionating a mixture to separate components thereof. In liquidchromatography, a sample containing a number of components to beseparated is injected into a fluid stream, and directed through achromatographic column. The column is designed to separate the mixturethrough differential retention on the column into component species. Thedifferent species then emerge from the column as distinct bands,separated in time.

A typical high performance liquid chromatography system (HPLC system)includes a pump for delivering fluids (the “mobile phase”) at acontrolled flow rate and composition, an injector to introduce a samplesolution into the flowing mobile phase, a tubular column encasementcontaining a packing material or sorbent (the “stationary phase”), and adetector to register the presence and amount of the sample components inthe mobile phase. When the mobile phase is passed through the stationaryphase, each component of the sample will emerge from the column at adifferent time because different components in the sample will havedifferent affinities for the packing material. The presence of aparticular component in the mobile phase exiting the column can bedetected by measuring changes in physical or chemical properties of theeluent. By plotting the detector's signal over time, response “peaks”corresponding to the presence of each of the components of the samplecan be observed and recorded.

A typical chromatography system includes a single column, or dimension,containing the stationary phase. One of the limits of traditionalchromatographic techniques is the limited number of components that canbe resolved in a single analysis. This limitation can be addressed usingmulti-dimensional chromatography. For example, in two-dimensionalchromatography, a particular group of components is transferred to asecond separation column. The group of components is typically one thatis not well separated on the first dimension, and may co-elute in asingle peak or band. The group of components, however, can be betterseparated on the second separation column. The second separation columntypically has an orthogonal mode of separation relative to the firstseparation column. However, all chromatographic techniques reach afundamental limit in the number of components that can be resolved in asingle analysis, and a given sample often has more than one component orgroup of components that requires this orthogonal separation.

Common practice has been to collect fractions from the separation foranalysis on a second system or a second series of runs with a differentmethod, or using the typical two-dimensional system. The sample isanalyzed repeatedly, as many times as the number of peaks required to becut. A different peak or time segment on each run is selected fortransfer to the orthogonal separation. Short cycle times on the seconddimension can also be used so that the two analyses remain coordinatedin parallel. This approach sacrifices resolution to save time.Additionally, analytical methods that incorporate separationtechnologies are required to resolve all sample components to provideunequivocal identification with the possibility of reliablequantitation, and all chromatographic techniques reach a fundamentallimit in the number of components that can be resolved in a singleanalysis.

Thus, improvements in efficiency, functionality, and accuracy ofmulti-dimensional liquid chromatography are needed in the art.

BRIEF SUMMARY OF THE INVENTION

The inventive disclosure is directed to new and useful chromatographysystems and methods for multidimensional chromatography. In variousembodiments of the inventive disclosure, the chromatography systemincludes a first chromatography column for receiving and separating aflow stream, a plurality of traps configured to trap a plurality ofdistinct flow segments exiting the first chromatography column duringseparation of the flow stream, and a second chromatography columnoperatively associated with the plurality of traps for receiving andseparating the distinct flow segments.

In certain embodiments, the chromatography system includes a pluralityof valves configured to selectively fluidly couple to one another, tothe first and second chromatography columns, and to the plurality oftraps. The plurality of valves may be configurable to a first positiondefining a first flow path which fluidly couples the firstchromatography column to a detector, and fluidly isolates the firstchromatography column from the plurality of traps, and a second positiondefining a second flow path which fluidly couples the firstchromatography column with a first of the plurality of traps fortrapping a first of the plurality of distinct flow segments, and fluidlyisolates the first chromatography column from all but the first of theplurality of traps. The plurality of valves may also be configurable toa third position defining a third flow path which fluidly couples thefirst chromatography column with a second of the plurality of traps fortrapping a second of the plurality of distinct flow segments, andfluidly isolates the first chromatography column from all but the secondof the plurality of traps.

In certain embodiments, the plurality of valves are configurable to afourth position defining a fourth flow path which fluidly couples thefirst trap and the second chromatography column to direct the firstdistinct flow segment from the first trap to the second chromatographycolumn, and a fifth position defining a fifth flow path which fluidlycouples the second trap with the second chromatography column to directthe second distinct flow segment to the second chromatography column.

The system may additionally include a plurality of pumps for pumping aplurality of flow streams through the chromatography system. Theplurality of pumps include a first pump in fluid communication with aninlet of the first chromatography column, and a second pump operativelyassociated with the second chromatography column and the plurality oftraps. In certain embodiments, the plurality of valves are configurableto a rinse position defining a sixth flow path which fluidly couples thesecond pump with the second chromatography column for rinsing the secondchromatography column, and fluidly isolates the second pump from theplurality of traps. The plurality of valves may also be configurable toa first release position which fluidly couples the second pump, thefirst trap, and the second chromatography column for releasing the firstdistinct flow segment from the first trap to the second chromatographycolumn, and fluidly isolates the second pump from all but the first ofthe plurality of traps. The plurality of valves can also be configuredto a second release position which fluidly couples the second pump, thesecond trap, and the second chromatography column for releasing thesecond distinct flow segment from the second trap to the secondchromatography column, and fluidly isolates the second pump from all butthe second of the plurality of traps.

In accordance with certain embodiments, the plurality of pumps caninclude a third pump, and in the second position of the plurality ofvalves, the third pump can be placed in fluid communication with thesecond flow path, and configured to dilute the first distinct flowsegment as the first distinct flow segment flows from the firstchromatography column to the first trap. In the third position of theplurality of valves, the third pump can be placed in fluid communicationwith the third flow path, and configured to dilute the second distinctflow segment as the second distinct flow segment flows from the firstchromatography column to the second trap. The plurality of valves canalso be configured to selectively fluidly couple the third pump to thereleased first distinct flow segment between the first trap and thesecond chromatography column, and to selectively fluidly couple thethird pump to the released second distinct flow segment between thesecond trap and the second chromatography column, such that the releasedfirst and second flow segments are weakened prior to reaching the secondchromatography column.

In accordance with additional embodiments, the plurality of traps caninclude at least one trap cartridge having absorbent material. In otherembodiments, the at least one trap cartridge can includes sixcartridges. In yet other embodiments, the plurality of traps can includeat least one empty tube.

The subject inventive disclosure is also directed to a chromatographysystem which includes a first chromatography column, a secondchromatography column, and a pair of valves having a plurality of ports.The pair of valves are configured to trap a plurality of distinct flowsegments which exit the first chromatography column during separation ofa flow stream therein between corresponding pairs of the plurality ofports, and to selectively fluidly couple each of the plurality oftrapped distinct flow segments with the second chromatography column. Incertain embodiments, each of the pair of multiport valves is a nineport, eight position valve.

The subject inventive disclosure is also directed to a chromatographymethod which includes directing a fluid sample through a firstchromatographic column configured to receive and separate the fluidsample, guiding a plurality of distinct flow segments exiting the firstchromatographic column during separation of the fluid sample in thefirst chromatographic column to a corresponding plurality of traps,trapping the plurality of distinct flow segments in the plurality oftraps, releasing the trapped plurality of distinct flow segments fromthe plurality of traps, and directing the released plurality of distinctflow segments through a second chromatographic column configured toreceive and separate the released plurality of distinct flow segments.In certain embodiments, the plurality of traps are operatively disposedbetween a pair of multiport valves, and the distinct flow segments ofthe separated fluid sample are fluidly coupled to the correspondingplurality of traps during different time segments.

The plurality of distinct flow segments of the separated fluid samplecan be trapped in the corresponding plurality of traps during a singleloading of the fluid sample through the first chromatographic column.Additionally, the plurality of distinct flow segments of the separatedfluid sample can be released from the plurality of traps and directedthrough the second chromatographic column in sequence. In accordancewith certain embodiments, each of the distinct flow segments can bediluted prior to trapping the plurality of distinct flow segments in theplurality of traps, and between the plurality of traps and the secondchromatographic column.

The subject inventive disclosure is also directed to a chromatographymethod which includes measuring a plurality of time segmentscorresponding to a plurality of peaks of a fluid sample flowing througha first chromatographic column configured to separate the fluid sample,and sequentially fluidly coupling a plurality of distinct flow segmentsexiting the first chromatographic column during separation of the fluidsample in the first chromatographic column with a correspondingplurality of traps. The sequential fluid coupling of the distinct flowsegments with the plurality of traps occurs during respective timesegments corresponding to the measured plurality of time segmentsassociated with the plurality of peaks. The distinct flow segments canbe trapped in the plurality of traps, sequentially released, and fluidlycoupled to a second chromatographic column. In certain embodiments, thedistinct flow segments are trapped in the plurality of traps during asingle loading of the fluid sample through the first chromatographiccolumn.

BRIEF DESCRIPTION OF THE DRAWINGS

So that those skilled in the art to which the systems and methods formultidimensional chromatography of the inventive disclosure appertainswill readily understand how to make and use the subject inventionwithout undue experimentation, preferred embodiments thereof will bedescribed in detail herein below with reference to certain figures,wherein:

FIG. 1 is an exemplary stacked configuration of a multi-dimensionalchromatography system in accordance with the inventive disclosure;

FIG. 2 is a schematic layout and exemplary flow diagram of amulti-dimensional chromatography system in accordance with the inventivedisclosure;

FIG. 3 is an enlarged view of the pair of multi-port valves and trapcartridges of FIG. 2;

FIG. 4 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a first position;

FIG. 5 is an exemplary chromatogram of a sample solution analyzed afterpassing through the first chromatography column, and highlights fourtime segments (cuts) corresponding to four exemplary peaks warrantinganalysis;

FIG. 6 is an exemplary chromatogram of the output of detector PDA-1 whenthe valves of the system are activated to divert flow to one or moretrap cartridges.

FIG. 7 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a second position;

FIG. 8 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a rinse position with atrapped flow segment;

FIG. 9 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a third position;

FIG. 10 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to an additional rinseposition

FIG. 11 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a fourth position; and

FIG. 12 is an exemplary flow diagram of the multi-dimensional system ofFIG. 2 illustrating the valves configured to a fifth position.

FIG. 13 is exemplary chromatogram showing the results of the seconddimension analysis with a trace of the mobile phase gradient as % B.

FIG. 14 is an exemplary chromatogram of a sample solution whichdemonstrates the identification of analytes in the second dimension.

FIGS. 15 and 16 are exemplary chromatographs showing the selectivitydifferences between the two dimensions of the multi-dimensional systemof FIG. 2.

FIG. 17 is a table showing the reproducibility of the retention timesfor four analytes using the methods of the claimed invention; and

FIG. 18 is an exemplary chromatogram showing the reproducibility ofretention times and the linear increase in peak size in the seconddimension in relation to sample injection size.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, new and useful chromatography systems andmethods for multidimensional chromatography are shown. Referring now toFIG. 1, an exemplary embodiment of the chromatography system of thepresent disclosure is shown. The system configuration is a three stacksystem. The first stack, shown on the left, includes the followingmodules from the bottom to top: QSM (First dimension pump), SM-FTN, andISM (Dilusion Pump). The second stack of the system, shown to the rightof the first stack, includes the following modules from bottom to top:BSM (Second dimension pump), CM, CM Aux, and two ACQUITY Divert Valveswith 6-port, two position valves. The Column Manager also contains two9-port, 8-position valves. The third stack of the system, shown to theright of the second stack, includes from bottom to top: a QDa (Seconddimension detector), a PDA-2 for the second dimension, and a PDA-1 forthe first dimension. The valves in the Column Manager are used to directflow through one of 6 trapping cartridges (or holding loops/bypasstubes), or to waste as further discussed below with respect to FIGS. 2-4and 7-12.

Referring now to FIGS. 2-3, a schematic layout and exemplary flowdiagram of the multi-dimensional chromatography system of FIG. 1 areshown, as well as an enlarged view of the pair of multi-port valves andtrap cartridges in accordance with the inventive disclosure. The systemallows for transfer of multiple components of a sample to a seconddimension, and for complete analysis of each of the multiple componentsin a single run, all in a fully automated system. The system can alsoinclude At-column dilution for adjusting the sample stream for optimaltrapping and improving second dimension analysis. By automating analysisof the components of a sample using two different chromatographicmechanisms, the inventive methodology of the present disclosure greatlyincreases selectivity and peak capacity, thus achieving completechromatographic resolution. Referring now to FIG. 4, themulti-dimensional system of FIG. 2 is shown with valves thereinconfigured to a first position. More particularly, the first position ofthe valves provides a flow path from the first dimension pump QSM,through the first chromatography column (Dimension 1) and the rightvalve VR, to the first detector PDA while the second dimension pump BSMis fluidly coupled to the left valve VL, the second chromatographycolumn (Dimension 2), and the second PDA-2 detector and the QDadetector. In this position, the flow path of the first dimension pump isfluidly isolated from the right valve VR and trap cartridges 1-6, andallows the first detector PDA to analyze the sample for peaks as itexits the first chromatography column in flow segments having differentcomposition based on the separation in the first chromatography column.If desired, the second dimension pump BSM can simultaneously be run torinse the second chromatography column.

Referring now to FIG. 5, an exemplary chromatogram of a sample solutionis recorded and analyzed while the sample passes through and isseparated by the first chromatography column. Four exemplary timesegments (cuts) corresponding to four exemplary peaks are deemed towarrant analysis, and are highlighted by the rectangular boxes shown inFIG. 5.

Referring now to FIG. 6, an exemplary chromatogram shows the output ofdetector PDA-1 when the valves of the system have been activated todivert the flow to one or more trap cartridges as discussed below.During those time segments, the effluent flow from the firstchromatography column is routed to the trap valve(s), and no flow goesto PDA-1. Thus, the peaks shown in FIG. 5 are not detected in FIG. 6.

Referring now to FIG. 7, the multi-dimensional system of FIG. 2 is shownwith valves therein configured to a second position. More particularly,the second position of the valves provides a flow path from the firstdimension pump QSM, through the first chromatography column, the rightvalve VR, the left valve VL, one of the pair of multi-port valves(bottom right at port 1), a first trap cartridge 1 disposed betweenports 1-1 of the pair of multi-port valves, the other of the pair ofmulti-port valves (bottom left at port 1), back to the left valve VL atport 3, and to waste while the second dimension pump BSM is fluidlycoupled to the left valve VL, the second chromatography column, and thesecond PDA-2 detector and the QDa detector. It will be appreciated thatgiven the sample data of FIG. 5, the system is configured to this secondposition at an elapsed time T1 corresponding to the beginning time ofthe first cut in FIG. 5 following loading of the sample into the firstchromatography column. For example, at time T1, a distinct flow segmentcorresponding to the first cut begins to exit the first chromatographycolumn, and the valves are set to provide the flow path of FIG. 7 sothat this first cut flow segment is routed to the first trap cartridgewhere a portion thereof is trapped. Any remaining flow is routed towaste through the left valve VL. It will be appreciated that during thisprocess, the valves can be configured as desired to trap distinct fluidsegments exiting the first chromatography column in any of the trapsdesired, and to fluidly couple and fluidly isolate various systemcomponents as needed.

Referring now to FIG. 8, the multi-dimensional system of FIG. 2 is shownwith the valves configured to a rinse position in which the distinctfirst cut flow segment corresponding to the first peak of the samplesolution is trapped in the first trap cartridge (e.g., between port 1 ofeach of the pair of multiport valves), the first dimension pump QSM isin fluid communication with the right valve VR and the first detectorPDA, and the second dimension pump BSM is fluidly coupled with the leftvalve VL, the second chromatography column, and the second PDA-2detector and the QDa detector. In this rinse position, the PDA isobserved until a time T2 corresponding to the beginning of the secondcut in FIG. 5, and the second chromatography column is again optionallyrinsed by the second dimension pump BSM.

Referring now to FIG. 9, at time T2, a distinct flow segmentcorresponding to the second cut begins to exit the first chromatographycolumn, and the valves are set to provide a flow path which routes thissecond cut flow segment to the second trap cartridge where a portionthereof is trapped. More particularly, this position of the valves shownin FIG. 9 provides a flow path from the first dimension pump QSM,through the first chromatography column, the right valve VR, the leftvalve VL, one of the pair of multi-port valves (bottom right at port 2),a second trap cartridge 2 disposed between ports 2-2 of the pair ofmulti-port valves, the other of the pair of multi-port valves (bottomleft at port 2), back to the left valve VL at port 3, and to waste whilethe second dimension pump BSM is fluidly coupled to the left valve VL,the second chromatography column, and the second PDA-2 detector and theQDa detector. In this manner this second cut flow segment is routed tothe second trap cartridge where a portion thereof is trapped, and anyremaining flow is routed to waste through the left valve VL.

Referring now to FIG. 10, the multi-dimensional system of FIG. 2 isshown with the valves configured to an additional rinse position inwhich the distinct first and second cut flow segments corresponding tothe first and second peaks of the sample solution are both trapped inthe first and second trap cartridges, respectively, the first dimensionpump QSM is in fluid communication with the right valve VR and the firstdetector PDA, and the second dimension pump BSM is fluidly coupled withthe left valve VL, the second chromatography column, and the secondPDA-2 detector and the QDa detector. In this rinse position, the PDA isobserved until a time T3 corresponding to the beginning of the third cutin FIG. 5.

The above described process is repeated, and the valves are switched toappropriate configurations (either manually or via automation), untilall of the distinct flow segments exiting the first chromatographycolumn corresponding to all of the cuts marked in FIG. 5 are captured inthe respective traps operatively disposed between the pair of multiportvalves. During the above described process, dilution pump ISM mayoptionally be utilized with a mixing ‘T’ in fluid communication with theflow paths to the respective traps in order to dilute (e.g., change theconcentration, pH, solvent identity, and/or concentration of strongsolvent) and thus better trap the distinct flow segments exiting thefirst chromatography column and routed to the traps in a way thatensures optimal retention on the next chromatographic element, by, forexample, changing pH or other parameter. The mixing ‘T’ configurationutilized can be in accordance with embodiments disclosed in, for exampleU.S. Pat. Nos. 6,790,361, 7,875,175, and 7,909,994, the disclosures ofwhich are hereby incorporated by reference in their entireties. ThisAt-column Dilution stage for adjusts the isolated peak compositionbefore the second mode of chromatography. While water may be utilized toreduce the organic solvent content, various techniques for pH adjustmentmay also be utilized. Various other dilution configurations andtechniques are described in U.S. application Ser. No. 62/286,603, thedisclosure of which is hereby incorporated by reference in its entirety.It will also be appreciated by those skilled in the art that theconfigurations of the valves described fluidly isolate as well asfluidly couple the trap cartridges, pumps, and chromatography columns asshown and as desired, and that additional configurations arecontemplated and within the scope of the inventive disclosure.

Referring now to FIG. 11, once all of the distinct flow segmentscorresponding to the peaks marked in FIG. 5 are trapped in therespective traps during a single loading of the sample through the firstchromatography column, the valves configured to a release position whichprovides a flow path from the second dimension pump BSM, through theleft valve VL, one of the pair of multi-port valves (bottom leftmultiport valve at port 1), the first trap cartridge, the other of thepair of multi-port valves (bottom right multiport valve at port 1), theleft valve VL again, the second chromatography column, and the seconddimension detectors while the first dimension pump is fluidly coupled tothe first chromatography column, the right valve VR, and the firstdetector. It will be appreciated that the second dimension pump BSM isthus used here to release the first cut distinct flow segment trapped inthe first trap cartridge (ports 1-1 of the multiport valves are opened,allowing release of the trapped flow segment through the center port ofthe bottom right multiport valve), and the valves are used to guide thereleased flow segment through the left valve VL and to the secondchromatography column for a second separation (which may be orthogonalto the first). Dilution pump ISM and mixing ‘T’ may similarly be usedhere to weaken the released flow segment prior to it reaching the secondchromatography column to improve separation functionality in the secondchromatography column. The segments passing through the secondchromatography column are observed with the second PDA-2 detector andthe QDa detector. Once this is completed, the valves can be configuredto allow the second dimension pump BSM to rinse the secondchromatography column.

Referring now to FIG. 12, the valves are configured to another releaseposition which provides a flow path from the second dimension pump BSM,through the left valve VL, one of the pair of multi-port valves (bottomleft multiport valve at port 2), the second trap cartridge, the other ofthe pair of multi-port valves (bottom right multiport valve at port 2),the left valve VL again, the second chromatography column, and thesecond PDA-2 detector and the QDa detector while the first dimensionpump QSM is fluidly coupled to the first chromatography column, theright valve VR, and the first detector PDA. Thus, it will be appreciatedthat the second dimension pump BSM is thus used here to release thesecond cut distinct flow segment trapped in the second trap cartridge(ports 2-2 of the multiport valves are opened, allowing release of thetrapped flow segment through the center port of the bottom rightmultiport valve), and the valves are used to guide the released flowsegment through the left valve VL and to the second chromatographycolumn for a second separation. Dilution pump ISM and mixing ‘T’ maysimilarly be used here to weaken the released flow segment from thesecond trap cartridge prior to it reaching the second chromatographycolumn to improve separation functionality in the second chromatographycolumn. This distinct fluid segment passing through the secondchromatography column is observed with the second PDA-2 detector and theQDa detector. Once completed, the valves can be configured to allow thesecond dimension pump BSM to rinse the second chromatography columnagain.

The above described process is repeated, and the valves are switched toappropriate configurations (either manually or via automation), untilall of the distinct flow segments trapped in the traps are released,routed to the second chromatography column, and analyzed. It will beappreciated that the systems and methodologies described hereineliminate the need for multiple runs to complete the analysis of severalcomponents within a sample, and can preserve full chromatographicresolution by allowing longer cycle times in the second dimension. Itwill also be appreciated that the above described systems and processesare exemplary embodiments of the inventive disclosure, and that thesystems and methodologies described herein can be utilized for anynumber of cuts and traps, including 2, 3, 4, 5, 6, 7, 8 . . . 16, etc.

In testing the above described system, trapping and the second dimensionanalysis were optimized with the use of At-column Dilution. Thechromatographic peaks in the second dimension were homogeneous, yieldingmuch easier spectral interpretation.

FIGS. 13-18 depict exemplary results of certain embodiments of thisdisclosure as appreciated by those having ordinary skill in the art inview of this disclosure.

Referring specifically to FIG. 14, the analytes as selected in FIG. 5were eluted from the high pH reversed phase column directly into theelectrospray source of the QDa. Each analyte was readily identifiedusing the SIR channels corresponding to the known components. The peaksin the final analysis are narrow and symmetrical, reflecting the usefuleffects of At column Dilution for re-focusing the analytes after eachchromatographic step. FIG. 15 shows the selection of a region of thechromatogram at low pH. This selected segment is transferred to thesecond dimension for chromatography at high pH. The orthogonal seconddimension provides resolution of coeluting peaks from the firstdimension. The peaks are also very sharp as a result of At-columnDilution between dimensions. In addition, the elution order issignificantly altered, reflecting the altered selectivity at the higherpH.

In preparation of FIGS. 1-18, the following instrumentation set up andsample protocols were used:

Columns:

-   -   First-Dimension: BEH C18 2.1×50 mm, 1.7 um,    -   Second Dimension: BEH C18 2.1×50 mm, 1.7 um,    -   Trapping column: XBridge C18 Direct Connect 2.1×30 mm,        Mobile phases:

QSM:

-   -   Solvent A: Water    -   Solvent B: Acetonitrile    -   Solvent C: 1% Formic Acid in Water

BSM:

-   -   Solvent A: 0.1% Ammonium Hydroxide in Water    -   Solvent B: 0.1% Ammonium Hydroxide in Acetonitrile

ISM: Water

Needle Wash: 80/20 ACN/water

Seal wash: 10% ACN

Injection volume: 2.0 μL

PDA:

-   -   Scan: 210 to 400 nm    -   Channel: 254 nm

QDa:

-   -   Scan: 100 to 650 Da        Temperatures:    -   Column 1: 40° C.    -   Column 2: 40° C.    -   Trap Column: Room Temperature        Samples:        Standards

Waters UPC2 Standard Mix:

-   -   2 mg/ml each of 3-benzoylpyridine, Cortisone, 4-nitroaniline,        4,4′-biphenol (in Methanol)

Waters Analgesic Mix Standard:

-   -   200 ug of each: Acetaminophen, Acetamidophenol, Acetanilde,        Acetylsalicylic acid, Caffeine, Phenacetin, Salicylic Acid, (in        Acetonitrile)

Waters ACQUITY/Quattro micro or Quattro Premier MS Start Up SolutionKit:

-   -   1.0 mg/mL of each: Sulfadimethoxine, Terfenadine, Reserpine,        Acetaminophen, and Caffeine (in Acetonitrile)

Final Sample Solution:

-   -   0.45 mg/mL Sulfadimethoxine    -   0.45 mg/mL Terfenadine    -   0.45 mg/mL Reserpine    -   0.45 mg/mL Acetaminophen    -   0.45 mg/mL Caffeine    -   90 ug/mL Acetamidophenol    -   90 ug/mL Acetanilde    -   90 ug/mL Acetylsalicylic Acid    -   90 ug/mL Phenacetin    -   0.20 mg/mL Salicylic Acid    -   0.20 mg/mL 3-benzoylpyridine    -   0.20 mg/mL Cortisone    -   0.20 mg/mL 4-nitroaniline    -   0.20 mg/mL 4,4′-biphenol        Protocol:

The sample is delivered to the first chromatographic column and elutedwith the first dimension mobile phase. At the time the desired peak isto be collected, the system valves are switched in order to direct thisportion of the sample from the first dimension to a sample loop or trapcartridge. While being transferred, the eluant is diluted with flow fromthe At-column dilution pump and the components from the first dimensionsegment are held in the loop or trap cartridge. The At-column dilutionfunction ensures that the selected sample components are retained as atight band at the entrance to the trapping cartridge. The diluent ischosen to increase retention and usually includes dilution with water,which may also adjust the pH or add ion pairing. Once the peak iscollected on the loop or cartridge, the valves switch back and the firstdimension separation continues. As soon as the next desired peak isreached on the first dimension, the valves are switched again in whichthe second sample component is collected in a different sample loop orcartridge. The process is repeated for the number of desired peaks to becollected from the sample. Once all the peaks have been collected on theloop or cartridges, the system valves are switched so that the seconddimension pump delivers the desired separation gradient of increasingorganic to elute the trapped analytes. As the analytes elute from thecartridge, they are diluted by the flow from the dilution pump. Thediluent is a composition that ensures the components are binding as anarrow band to the head of the second chromatographic column. Thegradient continues to elute the analytes from the second dimensioncolumn into the PDA and MS detectors.

Separation technology is fundamentally required to resolve all samplecomponents to provide unequivocal identification with usefulquantification in a single analytical method. All chromatographictechniques, however, reach a fundamental limit in the number ofcomponents that can be resolved in a single analysis. The abovedescribed system and methodology addresses this limitation.Additionally, traditional mobile phases for two chromatography modes areoften incompatible. Transferring a peak from one chromatographicseparation to another must avoid distortion of the injected peak thatwould compromise the separation on the second column. While this isoften achieved by transferring a very small volume, the presentdisclosure describes a combination of trapping and At-column Dilution totransfer the peak without constraining the volume, which preservesmaximum sensitivity of the analysis. In order to obtain the completebenefit of a second column, the modes of separation can be orthogonal toone another.

It is anticipated that the above described systems and methodologies canbe utilized for improved isolation in purification techniques and samplepreparation. The system can also be altered so that the sample isbrought to another analytical technique other than a Mass Spectrometer(e.g. NMR or a Fraction Collector).

It will be appreciated that various types of components may be utilizedfor the above described systems and methodologies. For example, thechromatography system described herein can also include any type ofchromatography technique that can be configured into a multi-dimensionalchromatography system, such as normal phase chromatography, reversedphase chromatography, carbon dioxide based chromatography, sizeexclusion chromatography, ion exchange chromatography, hydrophilicinteraction liquid interaction chromatography, hydrophobic interactionchromatography, affinity chromatography, and combinations thereof. Thechromatography system can also include various combinations oftechniques, such as reversed phase-reversed phase chromatography, normalphase-reversed phase chromatography, reversed phase-carbon dioxide basedchromatography, normal phase-carbon dioxide based chromatography, ionexchange-reversed phase chromatography, ion exchange-size exclusionchromatography, affinity chromatography-ion exchange, affinitychromatography-size exclusion, affinity chromatography-reversed phasechromatography. These combination techniques can be combined in anyorder.

The fluid pumps of the chromatography system described herein caninclude any pump capable of generating a fluid flow (e.g., flow stream)through the multi-dimensional chromatography system. Each fluid flow canindependently have a flow rate of about 0.01 uL/min, 0.1 uL/min, 1uL/min, 0.01 mL/min, 0.1 mL/min, 1 mL/min, 10 ml/min, 100 mL/min orabout 300 mL/min, depending on the chromatography techniques involved,the diameter of the tubing, valve orifices, column diameters, detectorcells, etc. These values can also be used to define a range, such asabout 0.01 to about 10 mL/min, or about 0.1 to about 2 mL/min.

Each fluid flow can independently contain various amount of organic,aqueous and compressible fluid (e.g., carbon dioxide) content. A fluidflow can contain about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or about 100% organic content. These valuescan be used to define a range, such as about 70% to about 90%. A fluidflow can also contain about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100% aqueous content. Thesevalues can be used to define a range, such as about 20% to about 40%. Afluid flow can contain about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100% compressible fluid(e.g., carbon dioxide) content. In one embodiment, a fluid flowcontaining carbon dioxide, e.g., a carbon dioxide based chromatographytechnique, can contain a co-solvent or modifier. The amount ofco-solvent or modifier in the carbon dioxide mobile phase can varydepending on whether it is organic or aqueous, as provided above. Theco-solvent or modifier can be methanol. In one embodiment, one of thefluid flows can be 95% carbon dioxide containing 5% methanol.

Each fluid flow can independently have various pH values. A fluid flowcan have a pH value of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5 and about 13. Thesevalues can be used to define a range, such as about 6 to about 8, orabout 3 to about 5. Each fluid flow can independently contain variousionic strength values of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 or about 5M. These values can be used to define a range, such as about 0.01 toabout 0.5 M. One or more of the fluid flows in the systems ormethodologies of the present disclosure can be a strong mobile phasewith respect to one of the chromatography techniques. A strong mobilephase is one that has a high elution strength and results in little orno retention of a component on a chromatography column, or sorbent. Asample or component dissolved in a strong mobile phase will have agreater affinity for the mobile phase than the stationary phase. One ormore of the fluid flows can be a weak mobile phase with respect to oneof the chromatography techniques. A weak mobile phase is one that has alow elution strength and results in high retention of a component on achromatography column, or sorbent. A sample or component dissolved in aweak mobile phase will have a lesser affinity for the mobile phase thanthe stationary phase.

As described herein, the chromatography system can include one or morechromatography columns, e.g., a first chromatography column, a secondchromatography column, etc. The columns can be any column used toseparate one or more analytes using one or more of the chromatographytechniques in a multi-dimensional chromatography system. The columns caninclude preparative columns, analytical columns and capillary columns.

The valves of the chromatography system can be any valve used with oneor more of the chromatography techniques and capable for diverting atleast one flow to at least two different flow paths. The valves can havemultiple ports and conduits, and be capable of diverting at least twoflows to at least two different flow paths wherein each can be divertedsimultaneously. The valves can also be capable of diverting at leastthree flows to at least two different flow paths wherein each can bediverted simultaneously (e.g., a 4 port valve, a 6 port valve, a 8 portvalve, a 10 port valve).

The detectors of the chromatography system can be any detector used withone or more of the chromatography techniques. The detector can be a UVdetector, a photo diode array detector, a mass spectrometer, a NMRdetector, a fluorescence detector, an evaporative light scatteringdetector, a charged aerosol detector, a conductivity detector, anelectrochemical detector or combinations thereof. In some embodiments,the present disclosure can incorporate traditionally non-compatibledetectors to the analysis of a sample by use of selectable At-ColumnDilution, such as by eliminating ion pairing reagents or adjusting pHfor best or consistent detection. The chromatography systems and methodsof the present disclosure can be used to transfer and interface withother analytical techniques, such as a fraction collector.

Some current chromatographic methods are not suitable for massdetection. These methods can be performed using the multi-dimensionalsystem of the present disclosure which can modify the method conditionsto be compatible with a mass detector. For example, the current methodcan be run on a first dimension in which a target peak is transferred toa second dimension. In the second dimension, the isolated peak can betrapped on a reversed-phase cartridge. After washing away the unretainedsalt and buffer, the isolated peak can be washed onto a secondreversed-phase column using a volatile mobile phase, e.g.,water-acetonitrile-formic acid. A gradient of these solvents can be usedto elute from the second reversed-phase column into the source of themass spectrometer. In the second dimension, the method conditions can bechanged in order to separate the target peak into individual componentsand at the same time obtaining conditions that are suitable for massdetection of the component(s) within the peak. Alternatively, the changein the second dimension can be a change in column chemistry, mobilephase, or combinations thereof.

The At-Column Dilution mixer (e.g., ACD “T”) of the chromatographysystem can include at least one capable of mixing two fluid flowstogether to form a combined fluid flow. The injector can be a fitting asdescribed in U.S. Pat. Nos. 6,790,361; 7,875,175 and 7,909,994, thecontents of each are incorporated herein by reference in theirentireties. The mixer can be a standard liquid chromatography tee, Y,inverted Y, or inverted Y union. The mixer can also be a smallpacked-bed mixer such as those used for solvent blending (e.g., WatersP/N 700002911).

The At-Column Dilution mixer can combine at least two flow streams,e.g., the first and second flow streams, having at least one physical orchemical difference between them to form a combined flow stream, e.g. athird flow stream. The mixer can combine the flow streams in any ratiofrom 0:1 to 1:0 to obtain a combined flow stream. The at least two flowstreams can have a physical or chemical difference between themincluding the ratio of organic/aqueous or carbon dioxide/organic/aqueouscontent, pH values, ionic strength values, or combinations thereof. Thecombined flow stream can be isocratic or can be a gradient with respectto one or more characteristics.

The system and method of the present disclosure can improve theinterface and transfer between at least two dimensions in amulti-dimension chromatography system by controlling or selectivelycombining at least two fluid flows (e.g., with the dilution pump) toform a combined flow having certain chemical or physical properties. Inone embodiment, the two fluid flows (e.g., a first and a second fluidstream) can be combined into a combined flow (e.g., a third fluidstream) wherein the combined flow stream has about 80%, 75, 70, 65, 60,55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or about 5%, or less (or more),by volume, of organic content or composition than one of the first flowstreams. These values can also be used to define ranges, such as about50% to about 20%. In particular, the two fluid flows can be combinedinto a combined flow wherein the combined flow has about 50% or less, byvolume, of organic content or composition than the first flow stream. Insome embodiments, the combined flow can have more, by volume, organiccontent. In other embodiments, the two fluid streams can be combinedinto a combined fluid stream wherein the combined flow stream has a pHvalue about or at least about, 0.5, 1, 1.5, 2, 2.5 or about 3 pH unitsdifferent (e.g., less or greater) than one of the first flow streams. Inparticular, the two fluid flows can be combined into a combined flowwherein the combined flow has a pH value about or at least about 1 pHunit different than the first flow stream.

In yet other embodiments, the two fluid streams can be combined into acombined fluid stream wherein the combined flow stream has about 80%,75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or about 5%, orless (or more) ionic strength than one of the first flow streams. Thesevalues can also be used to define ranges, such as about 60% to about30%. In particular, the two fluid flows can be combined into a combinedflow wherein the combined flow has about 50% or less ionic strength thanthe first flow stream. In some embodiments, the combined flow can havemore or a higher ionic strength.

The traps of the present system can include, for example, a loop, achromatography column, a cartridge, etc) to isolate one or more of thecomponents diverted from a first dimension separation. The trap(s) canhave a high affinity for one or more of the components diverted from afirst dimension separation. In one embodiment, the trap is capable ofphysically or chemically retaining an analyte in the third flow stream.The trap can be a trap column or cartridge containing a chromatographicmedia used for reversed phase, normal phase, affinity chromatography-ionexchange, affinity chromatography-size exclusion, affinitychromatography-reversed-phase, or combinations thereof. The traps caninclude one or more trap cartridges having absorbent material, but canadditionally or alternatively include one or more traps consisting of orcomprising an empty tube. One or more of the traps can contain a solidabsorbent or an empty tube that would hold the fluid segment from thefirst dimension in solution waiting for analysis in the seconddimension.

The systems and methods of the present disclosure can increase theretention of at least one diverted component (or analyte) on the trap(or other device) or additional chromatography column, e.g., the secondchromatography column. The retention of at least one the divertedcomponent can be increased by about 5%, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100% as compared to asystem or method without At-Column Dilution. These values can be used todefine a range, such as about 50% to about 90%.

The separation performance on the second chromatography column can beimproved by the system and method of the present disclosure. Forexample, the inclusion of the At-Column Dilution feature (e.g., the ISMDilution Pump) can increase the sensitivity, e.g., signal-to-noiseratio, of the one or more detectors downstream of the secondchromatography column by about 10%, 20, 30, 40, 50, 60, 70, 80, 90 orabout 95% or more compared to a system or method without At-ColumnDilution as described herein. The inclusion of the At-Column Dilutionfeature can also improve peak shape of components separated on thesecond chromatography column by about 10%, 20, 30, 40, 50, 60, 70, 80,90 or about 95% or more compared to a system or method without At-ColumnDilution as described herein.

While the subject inventive disclosure has been shown and described withreference to preferred embodiments, those skilled in the art willreadily appreciate that various changes and/or modifications may be madethereto without departing from the spirit and scope of the subjectinvention as defined by the appended claims.

The invention claimed is:
 1. A chromatography system, comprising: afirst chromatography column for receiving and separating a flow stream;a plurality of traps configured to trap a plurality of distinct flowsegments exiting the first chromatography column during separation ofthe flow stream; a second chromatography column operatively associatedwith the plurality of traps for receiving and separating the pluralityof distinct flow segments; and a plurality of pumps, one of theplurality of pumps operating as a dilution pump operative to provide afluid to dilute at least one of the plurality of distinct flow segments,wherein the dilution pump is configured to: dilute the at least one ofthe plurality of distinct flow segments as the plurality of distinctflow segments flow from the first chromatography column to the pluralityof traps, and dilute the at least one of the plurality of distinct flowsegments as the plurality of distinct flow segments flow from theplurality of traps to the second chromatography column.
 2. Achromatography system according to claim 1, further comprising: aplurality of valves configured to selectively fluidly couple to oneanother, to the first and second chromatography columns, and to theplurality of traps.
 3. A chromatography system according to claim 2,wherein the plurality of valves are configurable to a first positiondefining a first flow path which fluidly couples the firstchromatography column to a detector, and fluidly isolates the firstchromatography column from the plurality of traps, and a second positiondefining a second flow path which fluidly couples the firstchromatography column with a first of the plurality of traps fortrapping a first of the plurality of distinct flow segments, and fluidlyisolates the first chromatography column from all but the first of theplurality of traps.
 4. A chromatography system according to claim 3,wherein the plurality of valves are configurable to a third positiondefining a third flow path which fluidly couples the firstchromatography column with a second of the plurality of traps fortrapping a second of the plurality of distinct flow segments, andfluidly isolates the first chromatography column from all but the secondof the plurality of traps.
 5. A chromatography system according to claim4, wherein the plurality of valves are configurable to a fifth positiondefining a fifth flow path which fluidly couples the second trap withthe second chromatography column to direct the second distinct flowsegment to the second chromatography column.
 6. A chromatography systemaccording to claim 3, wherein the plurality of valves are configurableto a fourth position defining a fourth flow path which fluidly couplesthe first trap and the second chromatography column to direct the firstdistinct flow segment from the first trap to the second chromatographycolumn.
 7. A chromatography system according to claim 3, the at leastone pump comprising a plurality of pumps for pumping a plurality of flowstreams through the chromatography system, the plurality of pumpsincluding a first pump in fluid communication with an inlet of the firstchromatography column, and a second pump operatively associated with thesecond chromatography column and the plurality of traps.
 8. Achromatography system according to claim 7, wherein the plurality ofvalves are configurable to a rinse position defining a sixth flow pathwhich fluidly couples the second pump with the second chromatographycolumn for rinsing the second chromatography column, and fluidlyisolates the second pump from the plurality of traps.
 9. Achromatography system according to claim 7, wherein the plurality ofvalves are configurable to a first release position which fluidlycouples the second pump, the first trap, and the second chromatographycolumn for releasing the first distinct flow segment from the first trapto the second chromatography column, and fluidly isolates the secondpump from all but the first of the plurality of traps.
 10. Achromatography system according to claim 9, wherein in the secondposition of the plurality of valves, the dilution pump is in fluidcommunication with the second flow path, and configured to dilute thefirst distinct flow segment as the first distinct flow segment flowsfrom the first chromatography column to the first trap.
 11. Achromatography system according to claim 10, wherein the plurality ofvalves are configured to selectively fluidly couple the dilution pump tothe released first distinct flow segment between the first trap and thesecond chromatography column, and to selectively fluidly couple thedilution pump to the released second distinct flow segment between thesecond trap and the second chromatography column, such that the releasedfirst and second flow segments are diluted prior to reaching the secondchromatography column.
 12. A chromatography system according to claim 7,wherein the plurality of valves are configurable to a second releaseposition which fluidly couples the second pump, the second trap, and thesecond chromatography column for releasing the second distinct flowsegment from the second trap to the second chromatography column, andfluidly isolates the second pump from all but the second of theplurality of traps.
 13. A chromatography system according to claim 1,wherein the plurality of traps includes at least one trap cartridge withabsorbent material.
 14. A chromatography system according to claim 1,the at least one of the plurality of distinct flow segments dilutedprior to being trapped in the plurality of traps.
 15. A chromatographysystem according to claim 1, the at least one of the plurality ofdistinct flow segments diluted between the plurality of traps and thesecond chromatographic column.
 16. A chromatography method, comprising:directing a fluid sample through a first chromatographic columnconfigured to receive and separate the fluid sample; guiding a pluralityof distinct flow segments exiting the first chromatographic columnduring separation of the fluid sample in the first chromatographiccolumn to a corresponding plurality of traps; trapping the plurality ofdistinct flow segments in the plurality of traps; releasing the trappedplurality of distinct flow segments from the plurality of traps;directing the released plurality of distinct flow segments through asecond chromatographic column configured to receive and separate thereleased plurality of distinct flow segments; diluting at least one ofthe plurality of distinct flow segments via a dilution pump configuredto: dilute the plurality of distinct flow segments as the plurality ofdistinct flow segments flow from the first chromatographic column to theplurality of traps, and dilute the at least one of the plurality ofdistinct flow segments as the plurality of distinct flow segments flowfrom the plurality of traps to the second chromatographic column.
 17. Achromatography method according to claim 16, wherein the plurality oftraps are operatively disposed between a pair of multiport valves.
 18. Achromatography method according to claim 16, wherein the distinct flowsegments of the separated fluid sample are fluidly coupled to thecorresponding plurality of traps during different time segments.
 19. Achromatography method according to claim 18, wherein the plurality ofdistinct flow segments of the separated fluid sample are trapped in thecorresponding plurality of traps during a single loading of the fluidsample through the first chromatographic column.
 20. A chromatographymethod according to claim 18, wherein the plurality of distinct flowsegments of the separated fluid sample are released from the pluralityof traps and directed through the second chromatographic column insequence.
 21. A chromatography method according to claim 16, furthercomprising diluting each of the distinct flow segments prior to trappingthe plurality of distinct flow segments in the plurality of traps.
 22. Achromatography method according to claim 16, further comprising dilutingeach of the distinct flow segments between the plurality of traps andthe second chromatographic column.